Adaptation to hypoxia depends on the induction of a number of physiologically important genes such as erythropoietin, which regulates red blood cell mass, vascular endothelial growth factor, which promotes new blood vessel formation in ischemic tissue and tyrosine hydroxylase which is essential for the control of ventilation by the carotid body. The hypoxic up-regulation of these genes depends upon a common mode of oxygen sensing and signal transduction, leading to the activation of the transcription factor HIF-1. There is presumptive evidence that the oxygen sensor is a flavo-heme protein that functions as an NAD(P)H oxidase. In Specific Aim 1 of this proposal, a genetic strategy will be employed to clone and characterize genes that express proteins that have consensus NADPH and flavin binding domains. We have discovered three novel genes that have strong homology to cytochrome b5 reductase. One of these genes is of particular interest since it encodes a 488 residue fusion protein which, on the N-terminal side, bears homology to cytochrome b5 and, and on the C-terminal side, to cytochrome b5 reductase. This protein which we designate b5b5R3 is widely expressed in cells and tissues. The most challenging aspect of this proposal will be to determine whether b5b5R3, or the other two novel genes, b5R1 and b5R2, function as the oxygen sensor. Expression will be abolished by antisense experiments and, in the case of b5b5R3, by targeted knockout in mouse ES cells. Conversely, the functional properties of the proteins encoded by these three candidate genes will be tested by overexpression both in mammalian cells and in bacteria. Thorough spectroscopic analysis, including UV resonance Raman, will be accompanied by studies of enzymatic activity and substrate specificity. Specific Aim 2 describes a biochemical approach for characterization of two proteins isolated from the plasma membrane: a 50 kDa NADPH and flavin binding heme protein, that could be identical to b5b5R3, and a 240 kDa heme protein. If protein sequencing indicates that either protein is novel, it will be molecularly cloned and expressed, and its function will be assessed as described above. Specific Aim 3 focuses on the signal transduction process, determining the impact of the heme ligands carbon monoxide and nitric oxide on HIF-1 activation as well as the contribution of reactive oxygen species generated by the putative oxidase sensor. These experiments should provide a comprehensive body of information on the molecular events in the pathway that link a decrease in intracellular oxygen tension to transcriptional activation of biologically important genes.